The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW 264.7 cell culture system and assays for NF-B nuclear translocation, NF-B reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKLinduced NF-〉 activation. Introduction:The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. Materials and Methods: RANKL-induced RAW 264.7 cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-B nuclear translocation, NF-B reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-〉, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. Results: We found that TPA inhibited RANKL-induced RAW 264.7 cell differentiation into osteoclasts in a dosedependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-〉 activation in RAW 264.7 cells, but it suppresses the RANKL-induced NF-〉 activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-〉 activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-〉 complexes consisted of C-Rel, NF-B1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW 264.7 cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. Conclusion: Given that NF-B activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-〉 during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.
Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-B activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures.Introduction: Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-B activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-B activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption. Materials and Methods:We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-B luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW 264.7 cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained. Results:The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF-B activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-B activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes. Conclusions: Our data show that mutation of the p62 UBA domain results in increased activation of NF-B and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.
Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-B inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-B activity.Introduction: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-B activity. Materials and Methods:The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW 264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-B activation, p65 translocation, and IB-␣ degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6. Results: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (Ͻ1 M) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (Ͼ5 M) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-B activation, p65 translocation, and IB-␣ degradation both in mature osteoclasts and their precursors in a time-and dose-dependent manner. In addition, PAR inhibited NF-B activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1, or TNF-␣ to varying degrees and reduced the gene expression of RANK and TRAF6. Conclusion: The NF-B pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-B activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.
Bafilomycin A1, a specific inhibitor of V-ATPases, is a potent inhibitor of bone resorption, but the underlying mechanisms of its action remain unclear. In this study, we investigated the effect of Bafilomycin A1 on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts. Quantitative analysis by flow cytometry showed that Bafilomycin A1 increased total transferrin levels when RAW cells were exposed to labeled transferrin and decreased the total uptake of Dextran-rhodamine B, both in a dose- and time-dependent fashion, indicating that Bafilomycin influences receptor-mediated and fluid phase endocytosis in these cells. Furthermore, Bafilomycin A1 induced apoptosis of RAW cells in a dose dependent manner as evidenced by Annexin V flow cytometry. The action of Bafilomycin A1 on endocytotic events appeared to be more sensitive and occurred earlier than on its apoptosis inducing effects, suggesting that interrupting of endocytosis might be an early sign of Bafilomycin-mediated osteoclast inhibition. Semi-quantitative RT-PCR analysis showed that the gene transcripts of putative Bafilomycin A1 binding subunit, V-ATPase-subunit a3, were expressed in the preosteoclastic RAW cell line, and up-regulated during RANKL-induced osteoclastogenesis. Osteoclasts treated with Bafilomycin A1 exhibited apoptosis as well as altered cellular localization of Transferrin Alexa 647. Given that endocytosis and apoptosis are important processes during osteoclastic bone resorption, the potent effect of Bafilomycin A1 on endocytosis and apoptosis of osteoclasts and their precursor cells may in part account for Bafilomycin A1 inhibited bone resorption.
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